Conjugation of lipophilic albumin-binding moiety to rna for improved carrier-free in vivo pharmacokinetics and gene silencing

ABSTRACT

Provided herein are compounds and methods for gene silencing. The compound includes a RNA directly conjugated to an albumin-binding group. The method includes administering a compound comprising a RNA directly conjugated to an albumin-binding group to a subject in need thereof.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/371,619, filed Aug. 5, 2016, the entire disclosure of which isincorporated herein by this reference.

GOVERNMENT INTEREST

This invention was made with government support under grant numberR01EB019409 awarded by the National Institutes of Health (NIH), andgrant numbers 1349604, 1445191, 1445197, and 0909667 awarded by theNational Science Foundation (NSF). The government has certain rights inthe invention.

TECHNICAL FIELD

The presently-disclosed subject matter generally relates to products andmethods for RNA treatment and administration. More specifically, thepresently-disclosed subject matter relates to conjugated RNA compounds,methods of administering RNA compounds, and methods of treating diseaseswith RNA compounds.

BACKGROUND

Small interfering RNA (siRNA) has received significant attention inconnection with RNAi therapies due to its capacity to silencetraditionally undruggable targets. However, in vivo delivery barriershave typically limited clinical translation of siRNA, especially fornonhepatic targets such as solid tumors. More specifically, clinicaltranslation of therapies based on small interfering RNA (siRNA) has beenhampered by its comprehensively poor pharmacokinetic properties thatnecessitate molecule modifications and complex delivery strategies. Inparticular, systemic delivery of siRNA has been a challenge due to rapidrenal clearance from circulation, which leads to removal through theurine and provides only minimal bioavailability in target tissues. Thisrapid renal clearance limits the use of siRNA as a cancer therapeutic,as long vascular circulation time following intravenous injection is theprimary predictor of tumor biodistribution.

To overcome these delivery barriers, prior efforts have typicallyfocused on improving siRNA delivery through encapsulation innanoparticulate carrier systems. While a broad range of nanocarriersystems have shown the capacity to improve in vivo pharmacokinetics ofsiRNA, most of these carrier systems have not achieved clinicalrelevancy. This is due, in part, to their complex nature, preferentialuptake by clearance organs which limits delivery to target sites,variable distribution throughout target sites, and/or low therapeuticindices resulting from nonspecific, carrier-associated toxicities.

More recently, siRNA conjugates have emerged as an alternative tonanocarrier-mediated delivery, offering the possibility of improvingsiRNA pharmacokinetics without requiring a more complex deliveryvehicle. For example, Alnylam Pharmaceuticals has demonstrated high genesilencing potency of a trivalent N-Acetylgalactosamine (GalNAc) siRNAconjugate, which binds with high specificity and affinity to theasialoglycoprotein receptor on hepatocytes. Carrier-free gene silencinghas also been achieved in the liver with siRNA-cholesterol conjugates.However, siRNA conjugates that efficiently deliver to nonhepatic tissueshave yet to be developed.

Accordingly, there remains a need for methods and products that improvethe in vivo pharmacokinetics and gene silencing of siRNA, particularlyin nonhepatic tissue.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This Summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

In some embodiments, the presently-disclosed subject matter is directedto a compound comprising a RNA directly conjugated to an albumin-bindinggroup. In one embodiment, the albumin-binding group is hydrophobic. Inanother embodiment, the albumin-binding group is hydrophobic andanionic. In one embodiment, the albumin-binding group is a divalentlipidic moiety. In another embodiment, the divalent lipid moiety is adiacyl lipid. In a further embodiment, the divalent lipidic moietycomprises1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000] (L₂).

In some embodiments, the RNA is siRNA or miRNA. In one embodiment, thesiRNA comprises a functionalized siRNA. In another embodiment, thefunctionalized siRNA is functionalized with a dibenzocyclooctyne moiety.In some embodiments, the compound is complexed with albumin.

Also provided herein, in some embodiments, is a compound comprisingsiRNA functionalized with a dibenzocyclooctyne moiety and directlyconjugated to1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000].

Further provided herein, in some embodiments, is a method of genesilencing, the method comprising administering a compound comprising aRNA directly conjugated to an albumin-binding group to a subject in needthereof. In one embodiment, the albumin-binding group is hydrophobic. Inanother embodiment, the albumin-binding group is hydrophobic andanionic. In one embodiment, the albumin-binding group is a diacyl lipid.In one embodiment, wherein the albumin-binding group comprises1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000] (L₂). In some embodiments, the RNA is siRNA or miRNA. Insome embodiments, the siRNA comprises a functionalized siRNA.

In some embodiments, the administering comprises intravenousadministration. In some embodiments, the method further comprisestreating cancer in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E show graphs and images illustrating that successfullysynthesized and purified siRNA-L2 conjugate binds to albumin. (A)Abbreviated structures of reactants and final oligonucleotide-L2conjugate. Synthesis scheme is twostep; 1) siRNA-NH2+ DBCO-PEG4-NHSester, 2) siRNA-DBCO+DSPE-PEG(2000)-azide. (B) MALDI-TOF massspectrometry of the original amine-modified siRNA, the DBCOintermediate, and the L2 conjugate. (C) HPLC purification of siRNA-L2conjugate from reactant precursors. (D) L2 conjugation does not impactsiRNA silencing efficacy. A comparison of siRNA and siRNA-L2 silencingfrom in vivo jetPEI at a dose of 100 nM; n of 5, standard error shown.(E) Albumin binding measured by gel stained for siRNA (Top) and protein(Bottom). siRNA-L2 migrates as a micellar population alone andcomigrates with albumin, whereas unmodified siRNA does not migrate withalbumin. Note that albumin shows up as multiple bands due to running innondenaturing, native gel conditions.

FIG. 2A-B. (A) Critical micelle concentration of siRNA-L2 as determinedvia Nile Red assay. (B) Isothermal calorimetry (ITC) shows exothermicbinding of siRNA-L2 to bovine serum albumin (BSA) with a dissociationconstant of 1.38 μM.

FIGS. 3A-F. Show graphs and images illustrating that conjugation ofdiacyl lipid to oligonucleotides increases circulation half-life andreduces renal clearance. (A) Evaluation of association of siRNA/siRNA-L2with BSA or serum albumin in FBS by PAGE gel retardation assay. siRNA-L2alone (far right) migrates as a micellar population. Bound siRNA-L2migrates in the same location when mixed with BSA or FBS, suggestingthat siRNA-L2 is associating with the albumin component of FBS. Alsoshown are protein controls of BSA and FBS (left). (B) Cy5-labeledsiRNA-L2 and siRNA fluorescence in the blood measured in real timeintravitally by confocal microscopy after i.v. injection of CD1 mice.(C) Representative images of fluorescence in mouse blood vessels overtime. (D) siRNA-L2 shows association with albumin in vivo. Cy5-labeledsiRNA-L2 and siRNA was injected i.v. into CD1 mice and blood wascollected after 20 minutes. Serum isolated from blood components wasevaluated via PAGE gel retardation assay for the presence of Cy5-labeledoligonucleotide. Mice injected with siRNA had no Cy5 signal in theserum, but mice injected with siRNA-L2 showed faint bands correspondingto the unbound siRNA-L2 and a stronger band corresponding toalbumin-bound siRNA-L2. (E) siRNA and siRNA-L2 degrade over time in 60%FBS at 37o C. siRNA-L2 degrades more slowly than siRNA. (F) DNA, DNA-L2degrade on a similar time scale to siRNA. siRNA-L2. (G) Organbiodistribution of siRNA and siRNA-L2 at 20 min after i.v. injection.n=3, SE shown; ***P<0.001.

FIGS. 4A-E. Blood chemistry panel and body weight of mice injected withsiRNA-L2 (10 mg/kg) or in vivo jetPEI loaded with siRNA (1 mg/kg, 2mg/kg). (A) ALT: alanine aminotransferase; (B) AST: aspartateaminotransferase; (C) BUN: blood urea nitrogen; (D) Creatinine, (readingfor in vivo jetPEI at 2 mg/kg was not measurable). (E) Body weightpre-injection (day 0) and 24 hours post-injection (day 1). n=4, standarderror is plotted. 3 of 4 mice in the 2 mg/kg in vivo jetPEI did notsurvive treatment and could not be included in analysis.

FIGS. 5A-H siRNA-L2 achieves superior delivery to PDX and orthotopictumors. Biodistribution was evaluated using a nontoxic dose of 1, 10mg/kg of siRNA-L2 and the MTD of 1 mg/kg jetPEI NPs. (A-F) Orthotopicmodel: (A) Absolute organ radiance for siRNA-L2 (10 mg/kg), jet PEI NPs(1 mg/kg). (B) Fraction organ radiance for siRNA-L2, jetPEI NPs. (C)Absolute tumor radiance; exponential decay fits plotted. (D) Fractiontumor radiance; **P<0.01. (E) Tumor:liver ratio reveals a lowerproportion in the liver for siRNA-L2 in comparison with jetPEI NPs. n=4,SE plotted. (F) Representative images depicting accumulation in liver,tumors. (G and H) PDX model: (G) Biodistribution and (H) plotted tumorradiance (n=2) of dose-matched jetPET NPs and siRNA-L₂ at 24 h. Radianceunits are photons per

FIG. 6. Representative images of biodistribution to the organs inorthotopic tumor-bearing mice. siRNA-L2 was evaluated at 1, 10 mg/kg andjetPEI NPs were evaluated at 1 mg/kg.

FIGS. 7A-F. In an orthotopic tumor model, absolute radiance per eachorgan at (A) 30 minutes, (B) 24 hours, (C) 48 hours and fraction oftotal radiance per each organ at (D) 30 minutes, (E) 24 hours, (F) 48hours. n=4, standard error plotted.

FIGS. 8A-B. In a PDX tumor model, (A) absolute radiance per each organat 24 hours and (B) tumor:liver ratio of jetPEI NPs and siRNA-L2 in aPDX tumor model after intravenous injection at 1 mg/kg. n=2, standarderror plotted.

FIGS. 9A-D. siRNA-L2 penetrates tumors and is internalized by tumorcells, resulting in sustained gene silencing in a mouse tumor model. (A)Representative confocal microscopy images of tumor spheroid penetrationand internalization. (B) Cellular internalization of Cy5-labeledsiRNA-L2 or jetPEI NPs loaded with Cy5 siRNA in MCF-7 tumor spheroids,normalized to no treatment. Treatment at 100 nM, quantified by flowcytometry; n=3, SE plotted, ***P<0.001. (C) Cellular internalization intumor cells isolated from orthotopic xe-nograft mouse tumors afterinjection of jetPEI NPs at 1 mg/kg or siRNA-L2 at 1, 10 mg/kg,normalized to no treatment; n=6-8 tumors. (D) Gene silencing ofluciferase-targeted siRNA-L2 compared with unmodified siRNA in anortho-topic xenograft mouse tumor model; treatment at day 0 and 1 (asindicated by arrows) at 10 mg/kg, n=10. *P<0.05, **P<0.01: luc-L2 vs.scr-L2, ^(†)P<0.05, ^(‡)P<0.01: luc-L2 vs. luc. SE plotted.

FIGS. 10A-E. (A) Representative image of tumor spheroid uptake forsiRNA. (B) Cellular uptake, as evaluated by flow cytometry, of MCF-7breast cancer cells grown in tumor spheroids. Data are expressed as foldincrease in fluorescence relative to untreated cells. Treatment with invivo jetPEI complexes resulted in significantly less uptake than siRNA,while siRNA-L2 achieved the highest uptake. (C) Percentage positivecells, as evaluated by flow cytometry, of MCF-7 breast cancer cellsgrown in tumor spheroids. Treatment with in vivo jetPEI complexesresulted in significantly fewer positive cells than siRNA and siRNA-L2,consistent with its poor penetration into the interior of the tumorspheroids. (D) Representative histograms of flow cytometric evaluationof Cy5-labeled siRNA uptake by MCF-7 breast cancer cells grown in tumorspheroids. (E) Percentage cy5 siRNA positive tumor cells isolated fromorthotopic xenograft mouse tumors. n=6 to 8. n=3, standard errorplotted; **=p<0.01, ***=p<0.001.

FIGS. 11A-D. (A) Representative images of tumor luminescence in micewith orthotopic luciferase-expressing tumors, treated with luc-L2, luc,or scr-L2 at 10 mg/kg on day 0 and 1. (B) Gene silencing jetPEI NPscomplexed with luc compared to scr siRNA in an orthotopic xenograftmouse tumor model; treatment at day 0 and 1 (as indicated by arrows) at1 mg/kg, n of 8; *=p<0.05. (C) Mouse body weight after treatment withluc-L2, luc, or scr-L2 at 10 mg/kg on day 0 and 1; body weight isconsistent across treatment groups over the course of the experiment.n=5. (D) Mouse body weight after treatment jetPEI NPs complexed with lucor scr siRNA at 1 mg/kg on day 0 and 1; body weight is consistent acrosstreatment groups over the course of the experiment. n=5. Standard erroris plotted for all.

FIGS. 12A-B. siRNA-L2 (A) silences therapeutic gene MCL-1 and (B)increases caspase activity in vitro. Treatment of MCF-7 cells at 200 nMfor 24 hours in 10% serum; n=3, standard error plotted. ***p<0.01.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

While the terms used herein are believed to be well understood by thoseof ordinary skill in the art, certain definitions are set forth tofacilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong.

All patents, patent applications, published applications andpublications, GenBank sequences, databases, websites and other publishedmaterials referred to throughout the entire disclosure herein, unlessnoted otherwise, are incorporated by reference in their entirety.

Where reference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, Biochem. (1972)11(9):1726-1732).

Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are described herein.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, the term “small interfering RNA” or “siRNA” refers to adouble-stranded RNA molecule, 20-25 base pairs in length, withphosphorylated 5′ ends and hydroxylated 3′ ends including twooverhanging nucleotides. siRNA interfere with the expression of geneshaving complementary nucleotide sequences by degrading mRNA aftertranscription.

As used herein, the term “microRNA” or “miRNA” refers to ribonucleicacid sequences that when made synthetically are structurally the same assiRNA, but are synthesized based on a sequence that is expressed in vivorather than a sequence that is “discovered” and fully synthetic likesiRNA.

The presently-disclosed subject matter includes methods and products foradministration of RNA. In one embodiment, the product includes an RNAconjugate. In another embodiment, the RNA conjugate includes a RNAmolecule directly conjugated to an albumin-binding group.

The RNA molecules include any suitable double-stranded RNA molecules,such as, but not limited to, small interfering RNA (siRNA) and/ormicroRNA (miRNA). For example, in one embodiment, the albumin-bindinggroup may be conjugated to the 3′ end of the passenger strand of anysiRNA and/or miRNA molecule. Although described generally with respectto conjugation of the 3′ end of the passenger (sense) strand, as will beappreciated by those skilled in the art, the disclosure is not solimited and includes conjugation to other termini such as the 5′ end ofthe passenger strand, the 3′ end of the guide/antisense strand, or the5′ end of the guide/antisense strand.

The albumin-binding groups include, but are not limited to, any suitablehydrophobic and/or anionic compound. Suitable albumin-binding groups mayinclude hydrophobic molecules and/or lipids, such as palmitate; monacylchains of varied lengths; diacyl chains of varied lengths; chains havingvaried levels of saturated (double) bonds; diacids; aromatic ligands;heterocyclic ligands, such as evans blue; anionic molecules; moleculesthat are both anionic and hydrophobic, including anionic and hydrophobicpeptides; aptamers; or a combination thereof. For example, in oneembodiment, the RNA is directly conjugated to a divalent lipidic moiety.In another embodiment, the RNA is directly conjugated to a C18 diacyllipid. In a further embodiments, where the albumin-binding groupincludes a diacid, the hydrophobic chain thereof has a carboxylic acidend functionality that improves albumin binding.

In one embodiment, the RNA molecule is conjugated to the albumin-bindinggroup through one or more crosslinkers. In another embodiment, using oneor more of the crosslinkers disclosed herein facilitates conjugation ofany suitable RNA molecule to any suitable albumin-binding group. Thecrosslinker(s) include any suitable linker capable of coupling the RNAto the albumin-binding group. Suitable crosslinkers include, but are notlimited to, heterobifunctional linkers, polyethylene glycol (PEG)linkers having a molecular weight of between 0 and 40,000, peptidelinkers, or a combination thereof. For example, suitable PEG crosslinkerinclude PEG molecules having 45 repeat units (i.e., a molecular weightof about 2000 g/mol), 24 repeat units, 12 repeat units, 6 repeats, anyother suitable number of repeat units providing a molecular weightwithin the range disclosed herein, or any combination, sub-combination,range, or sub-range thereof. Other suitable non-PEG crosslinkersinclude, for example, cleavable peptide linkers, such as cathepsin,which promote release of siRNA for albumin binder in the endosome;peptides with endosome disruptive function, such as melittin; peptidesthat serve as a simple spacer, such as polyglycine; peptides thatcooperate with the albumin-binding group to improve albumin binding,such as negatively charged and hydrophobic amino acid containingsequences; cell penetrating peptides that promote cell uptake of thecargo, such as the CPP “Tat” or poly(arginine); or a combination thereof

In some embodiments, the RNA is functionalized and/or modified prior toconjugation. In one embodiment, modifying and/or functionalizing the RNAincludes attaching a linker thereto. In another embodiment, the linkeris attached through any suitable method, including, but not limited to,“click” (octyne-azide) chemistry, N-hydroxysuccinimide (NHS)-aminereaction, thiol-maleimide reaction, guest-host interaction (e.g.,biotin-streptavidin or cyclodextrin-adamantane), or a combinationthereof. In a further embodiment, attaching the linker includes firstmodifying the RNA with an amine, then reacting the amine modified RNAwith the linker. For example, attaching a N-hydroxysuccinimide (NHS)ester/octyne linker to an siRNA may include modifying the siRNA with anamine, and subsequently reacting the amine-modified siRNA with adibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester.

Additionally or alternatively, the albumin-binding group may be modifiedand/or selected to bind the RNA and/or functionalized RNA to form theRNA conjugate. For example, in one embodiment, albumin-binding groupincludes or is modified to include an azide functional diacyl moleculethat is arranged and disposed to react with an octyne on thefunctionalized RNA. In a more specific example, the functionalized RNAis directly conjugated to1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000] (DSPE-PEG₂₀₀₀-azide, or L₂) to form the RNA conjugate,termed siRNA-L₂. Although described above with regard to an azidefunctional diacyl reacting with an octyne, as will be appreciated bythose skilled in the art, the disclosure is not so limited and mayinclude other methods of conjugation including the use of other reactivegroups on the albumin binder, such as direct use of an amine orcarboxylic acid for attachment.

In some embodiments, the siRNA conjugate, such as siRNA-L₂, binds theserum protein albumin via interaction with a two-tailed lipid chain. Inone embodiment, the binding of the siRNA conjugate to the serum proteinalbumin enhances the pharmacokinetic properties of the siRNA as comparedto an unmodified siRNA and/or existing nanocarrier. In anotherembodiment, enhancing the pharmacokinetic properties includes increasingthe circulation half-life and/or bioavailability of the siRNA, ascompared to existing nanocarriers and/or unmodified siRNA. Additionallyor alternatively, enhancing the pharmacokinetic properties may includeincreasing the quantity of cellular or tumor accumulation, increasingthe homogeneity of cellular or tumor accumulation, increasing resistanceto nucleases, and/or permitting increased dosing amount with decreasedtoxicity as compared to unmodified siRNA and/or existing nanocarriers.

For example, in one embodiment, in contrast to existing nanocarrieswhich provide a tumor:liver accumulation ratio of less than 3:1, theconjugation of L₂ provides a tumor:liver accumulation ratio of at least5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, or anycombination, sub-combination, range, or sub-range thereof. In anotherembodiment, the siRNA-L₂ conjugate provides uptake in at least at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, and/or at least 99% of tumor cells.

In certain embodiments, the conjugation of L₂ provides at least a 4-foldincrease in circulation half-life, at least a 6-fold increase inbioavailability, and/or decreased renal accumulation as compared tounmodified siRNA and/or existing nanoparticle carriers. For example, inone embodiment, the conjugation of L₂ increases circulation half-life atleast 7.5-fold and/or bioavailability at least 13-fold as compared tounmodified siRNA. In another embodiment, the conjugation of L₂ increasestumor accumulation at least 19-fold and/or per-tumor-cell uptake atleast 46-fold as compared to existing siRNA nanocarriers.

In addition to enhancing the pharmacokinetic properties as discussedabove, the conjugation of siRNA to a divalent lipidic moiety maintainsor substantially maintains the gene silencing activity of the conjugatedsiRNA. Accordingly, in some embodiments, the siRNA conjugate providesincreased gene silencing as compared to unmodified siRNA and/or existingnanocarriers. Without wishing to be bound by theory, it is believed thatthe increased circulation time, tumor penetration, and tumordistribution of the siRNA conjugates, along with the maintained orsubstantially maintained gene silencing activity, facilitate use of theconjugate as a cancer therapeutic.

Also provided herein, in some embodiments, is a method of genesilencing. In one embodiment, the method includes administering one ormore of the siRNA conjugates disclosed herein to a subject in needthereof. The siRNA conjugates may be administered by any suitableprocess, including, but not limited to, intravenous (i.v.)administration. In another embodiment, after administration, the siRNAconjugates bind to endogenous albumin, which provides the enhancedpharmacokinetics discussed herein. Additionally or alternatively, atleast one of the siRNA conjugates may be pre-complexed with albuminprior to administration. In a further embodiment, the siRNA conjugatesare administered without a nanocarrier or other encapsulation/carriercompound.

In certain embodiments, because albumin is not trafficked to the liver,the siRNA conjugate mediates gene silencing at alternative targets whereaccumulation occurs as a result of long circulation time. In oneembodiment, the alternative target is a tumor. For example, siRNA-L₂mediates enhanced gene silencing in vitro in MDA-MB-231 breast cancercells compared to unmodified siRNA. Accordingly, in some embodiments,the method of gene silencing further includes treating a disease in asubject in need thereof. The disease includes any disease state that canbe treated through effective i.v. RNAi therapy. For example, onetherapeutic aim includes the treatment of tumors. In another example,the increased vascular permeability of the tumors leads to increasedaccumulation of long-circulating entities having a size of less thanabout 200 nm, less than about 100 nm, less than about 50 nm, less thanabout 25 nm, less than about 20 nm, less than about 15 nm, less thanabout 10 nm, less than about 9 nm, less than about 8 nm, less than about7 nm, about 7 nm, or any combination, sub-combination, range, orsub-range thereof.

In some embodiments, the siRNA conjugate is modified to provide and/orfacilitate targeting siRNA-L₂ to a particular desired cell type orphysiological site and/or to mediate endosomal escape. Additionally oralternatively, the delivery strategy of the siRNA conjugate may bemodified to provide and/or facilitate targeting siRNA-L₂ to a particulardesired cell type or physiological site and/or to mediate endosomalescape.

EXAMPLES Example 1

Significance

This example describes a diacyl lipid-modified siRNA that leveragesalbumin as an endogenous carrier, resulting in comprehensively enhancedpharmacokinetic properties that translate to greater quantity andhomogeneity of tumor accumulation relative to nanocarriers. Thealbumin-binding siRNA conjugate strategy described herein issynthetically simple and safe at high doses, and thus is a translatableand potentially transformative option for RNAi oncology therapies.

The enhanced permeability and retention (EPR) effect, based upon thehigh vascular permeability and diminished lymphatic drainage at tumorsites, suggests a preferential tumor accumulation of particles ofnanocarrier size (˜100 nm). However, the EPR phenomenon as a tumortargeting strategy has recently come under intense scrutiny due to thediscrepancy observed between preclinical and clinical efficacy ofnanoparticle-based cancer therapeutics. There is a growing appreciationthat among wildly heterogeneous human cancers, the EPR effect may beonly relevant in select tumor or patient subsets. In particular, thewidespread “leakiness” of tumor vasculature, a characteristic of rapidlydeveloping mouse tumor models, has likely been exaggerated in itsrelevance to slower-forming human lesions. The field of nanomedicine hasresponded to these realizations with efforts to enhance understanding ofnanoparticle performance in animal models, strategies to normalize tumorvasculature, systematic investigations into ideal nanoparticlecharacteristics, and a focus on smaller (20-30-nm-sized) nanocarriers.Despite the promise of these approaches, the diversity of human cancersnecessitates equivalently diverse delivery approaches and opportunityfor improvement remains, particularly in the area of enhancinguniformity of tumor distribution.

As an alternative approach to commonly used nanoparticle carriers, theinstant inventors leveraged the long-lived endogenous serum proteinalbumin as an siRNA carrier. This example describes the synthesis ofsiRNA conjugated to a diacyl lipid moiety (siRNA-L₂), which rapidlybinds albumin in situ. In comparison with unmodified siRNA, siRNA-L₂exhibited a 5.7-fold increase in circulation half-life corresponding toan 8.6-fold increase in bioavailability and reduced renal accumulation.Benchmarked against leading commercial siRNA nanocarrier in vivo jetPEI,siRNA-L₂ achieved 19-fold greater tumor accumulation and 46-foldincrease in per-tumor-cell uptake in a mouse orthotopic model of humantriple-negative breast cancer.

Additionally, in contrast to nanoparticles that typically exhibitconcentration of dose near leaky vessels but not within more avasculartumor regions, resulting in inhomogeneous efficacy and higher potentialfor incomplete remission and recurrence, the smaller, long-circulatingsiRNA conjugates provide an alternative that creates more homogeneoustherapeutic distribution within tumors. Indeed, the apparent tissuepermeability of the serum protein albumin [hydrodynamic size ˜7.2 nm] isconsistently more than fourfold greater than that of 100-nm liposomes ina variety of mouse models of breast cancer. With respect to the instantconjugates, siRNA-L₂ penetrated tumor tissue rapidly and homogeneously;30 min after i.v. injection, siRNA-L₂ achieved uptake in 99% of tumorcells, compared with 60% for jetPEI. Remarkably, siRNA-L₂ achieved atumor:liver accumulation ratio >40:1 vs. <3:1 for jetPEI. The improvedpharmacokinetic properties of siRNA-L₂ facilitated significant tumorgene silencing for 7 d after two i.v. doses.

Proof-of-concept was extended to a patient-derived xenograft model, inwhich jetPEI tumor accumulation was reduced fourfold relative to thesame formulation in the orthotopic model. The siRNA-L2 tumoraccumulation diminished only twofold, suggesting that the superior tumordistribution of the conjugate over nano-particles will be accentuated inclinical situations. These data reveal the immense promise of in situalbumin targeting for development of translational, carrier-freeRNAi-based cancer therapies. In particular, it is anticipated thatalbumin-associated siRNA will show promise as a cancer therapeutic byextending the circulation time of siRNA, enabling efficient tumor tissuepenetration, and leveraging the propensity of tumor cells to internalizealbumin.

In situ targeting of albumin following i.v. delivery is a viablestrategy because endogenous albumin is the most abundant serum protein(>40 mg/mL) and has a circulation half-life of about 20 d. It is also anatural carrier of and has a high affinity for poorly soluble lipids.Although previous work has established the utility of interaction ofhigh-and low-density lipoproteins with cholesterol-conjugated siRNA, thenatural trafficking of these lipoproteins concentrates the therapy inthe hepatocytes of the liver. The potential of albumin-bound siRNA hasbeen minimally explored. In the unique strategy discussed herein, thecapacity of albumin to bind fatty acids is exploited by modifying siRNAwith a lipidic moiety designed for high-affinity albumin binding.Through modification of siRNA with a lipidic albumin-targeting agentrather than alternative albumin-binding molecules like peptide domainsand a truncated Evans blue, the hydrophobically modified siRNA exhibitedimproved resistance to nucleases and enhanced cellular internalization.Thus, the strategic choice of modification with an albumin-binding lipidhas the potential to confer additional advantages in siRNA stability andcell membrane interactions for uptake and endosomal escape in additionto circulation persistence, tissue penetration, and biodistribution. Toinvestigate the clinical potential of the siRNA conjugate, its efficacywas examined as a systemic RNAi cancer therapeutic by evaluatingdelivery and gene silencing in translationally relevant models of humantriple-negative breast cancer.

Results

Purified siRNA-L₂ Conjugate Binds to Albumin. To synthesize siRNA-L₂, asingle-stranded amine-modified siRNA was reacted with an NHSester/octyne heterobifunctional cross-linker and subsequently conjugatedwith1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000] (DSPE-PEG2000-azide) to generate siRNA-L₂ (FIG. 1A). Thefully purified L₂ conjugates were obtained by reverse-phasechromatography and purity was confirmed by mass spectrometry (FIGS.1B-C). Following purification, sense strand siRNA-L₂ was annealed to thecorresponding antisense strand (for imaging studies, the antisensestrand was Cy5-labeled). It was confirmed that conjugation of the L₂moiety to siRNA did not significantly impact its inherent gene silencingactivity, as demonstrated by in vitro knockdown evaluation of siRNA andsiRNA-L₂ delivered via the commercial transfection reagent in vivojetPEI (FIG. 1D).

The albumin-binding capacity of siRNA-L₂ was confirmed using anondenaturing, native PAGE assay (FIG. 1E). siRNA-L₂ alone migratesabove the albumin band because it exists as a micellar population at theconcentration loaded into the gel (0.05 mg/mL), whereas critical micelleconcentration is 1.4 μg/mL (FIG. 2A). As the albumin:siRNA-L₂ ratioincreases, more siRNA-L₂ binds to and migrates with albumin. UnmodifiedsiRNA does not bind to albumin to any degree at any of theconcentrations tested. Evaluation of siRNA-L₂ binding to albumin viaisothermal calorimetry further confirmed spontaneous association of themolecules (dissociation constant was 1.38 μM; FIG. 2B). Binding of L₂conjugates to albumin in the presence of complete serum was alsoevaluated by gel migration assay, revealing preferential binding to thealbumin component of serum (FIG. 3A).

Albumin Binding of siRNA-L2 Enhances Circulation Time and Reduces RapidRenal Clearance. To characterize the in vivo pharmacokinetics ofsiRNA-L₂ in comparison with unmodified siRNA, circulation persistencewas evaluated in real time using intravital confocal microscopyfollowing i.v. injection (Materials and Methods). The circulationhalf-life_((1/2)) of siRNA-L2 was 5.7-fold longer than unmodified siRNA(FIGS. 3B-C and Table 1). Additionally, the area under the curve, ameasure of bioavailability of systemically delivered therapeutics, was8.6-fold greater for the L₂-conjugate compared with unmodifiedoligonucleotide. To evaluate in situ albumin binding, serum samples frommice injected with siRNA-L₂ (blood collection at 20 min postinjection)were evaluated via PAGE gel migration assay and revealed the presence ofalbumin-bound siRNA-L₂ (FIG. 3D). These data confirm that albumin actsas a chaperone for siRNA-L₂ in vivo and establish that siRNA-L₂association with albumin confers significant improvements in siRNApharmacokinetics. To support these studies, the time scale ofdegradation of unmodified and L₂-modified oligonucleotides wasinvestigated. siRNA and siRNA-L₂ showed resistance to degradation inserum over the pharmacokinetic time frame assessed, and L₂ modificationimparted a slight improvement in resistance to serum degradation (FIG.3E-F).

TABLE 1 Key pharmacokinetics parameters for siRNA-L₂ vs. siRNA ParametersiRNA siRNA-L₂ P value t½, circulation, min 2.3 ± 0.2 13.1 ± 1.6 0.0023AUC_(circ, 0-∞), fluor. 5,500 ± 800   47,300 ± 6,700 0.0034 intensity ×min Fraction kidney 0.790 ± 0.018  0.503 ± 0.014 <0.0001 radiance

Biodistribution of siRNA vs. siRNA-L₂ was evaluated in excised organs at20 min postinjection. For in vivo studies, siRNA-L₂ exhibited increasedaccumulation in almost all organs, likely due to its prolongedcirculation time and reduced clearance into the urine in comparison withunmodified siRNA (FIG. 3G). The kidneys were the sole exception, showingsignificantly more unmodified siRNA accumulation (a 1.6-fold greaterfraction of the total organ radiance) at this early time point. Thisillustrates that using albumin as a natural carrier for siRNA-L₂ allowsreduction of acute clearance through the renal route.

siRNA-L₂ Outperforms a Leading in Vivo Nanoparticle Carrier in Safetyand Tumor Accumulation. The reduction in kidney accumulation andprolonged circulation half-life of siRNA-L₂ motivated a comparison withcommercially available in vivo nanoparticles. Of particular interest isthe biodistribution profile of siRNA-L₂ in comparison with typicalnanocarriers, as high uptake by mononuclear phagocytic system organs(the liver and the spleen) can result in minimal dose accumulation atthe target site. Compared with nanoparticles, siRNA-L₂ is expected toavoid this off-target accumulation and to more readily penetrate tumortissue.

siRNA-L₂ was compared with a leading formulation for nanoparticle-basedin vivo nucleic acid delivery, in vivo j etPEI. In vivo jetPEInanoparticles (jetPEI NPs) have been used in clinical trials, and thiscomparison is therefore a stringent test for therapeutic potential.Before in vivo biodistribution studies, tolerated doses were determinedfor siRNA-L₂ and jetPEI NPs. siRNA-L₂ is expected to avoid the toxicside effects associated with high doses of cationic nanocarriers,permitting safe use at higher dosages and potentially expanding theultimate therapeutic index of siRNA drugs. Toxicity was investigated bymonitoring mouse body weight and quantifying blood chemistry markers ofliver [alanine aminotransferase (ALT) and aspartate aminotransferase(AST)] and kidney [blood urea nitrogen (BUN) and creatinine] toxicity.Mice injected with an siRNA-L₂ dose of 10 mg/kg exhibited normal ALT,AST, and BUN levels statistically equivalent to those of saline-injectedmice; these mice also showed no change in body weight (FIGS. 4A-E).Delivery of jetPEI NPs at a dose of 1 mg/kg created no signs oftoxicity, but doubling that dose to 2 mg/kg resulted in mortality forthree of four mice and showed marked hepatic and renal toxicity in thesingle surviving mouse. These data suggest that siRNA-L₂ is a saferalternative to nanocarrier-based delivery with the potential for a muchbroader therapeutic index. The maximum tolerated dose (MTD) of 1 mg/kgfor in vivo jetPEI and a well-tolerated dose of 10 mg/kg for siRNA-L₂were used in subsequent studies (MTD not determined for siRNA-L₂).

The biodistribution profile of the L₂ conjugate vs. jetPEI NPs wasevaluated in a mouse orthotopic xenograft tumor model. siRNA-L₂ orjetPEI NPs were injected i.v. into tumor-bearing mice and organs wereevaluated for siRNA accumulation. Comparing the absolute radiance in theorgans over time from mice treated with jetPEI NPs or siRNA-L₂, it isevident that the 10-mg/kg treatment of siRNA-L₂ significantly enhancesaccumulation in all of the organs at an acute (30 min) time point (FIGS.5A, 6, and 7A). Notably, the vast majority of siRNA-L₂ was cleared fromall organs excepting the kidneys and tumors by 24 h (FIGS. 5A and 7A-F).jetPEI NPs, in contrast, create higher proportional delivery to andretention within the mononuclear phagocyte system (MPS) clearance organs(the liver and spleen) (FIG. 5B).

The in vivo tolerability of high siRNA-L₂ doses enables a remarkableincrease in tumor accumulation (FIGS. 5C-D). The area under the curvewithin the tumor was 19.3-fold higher for siRNA-L₂ at 10 mg/kg than forthe maximum tolerated dose of jetPEI NPs (Table 2). Dose-matchedsiRNA-L₂ at 1 mg/kg also outperforms jetPEI NPs in this measure of tumoraccumulation by 2.4-fold. Additionally, the fraction of the total organradiance in the tumors is consistently higher for siRNA-L₂ at both dosescompared with jetPEI NPs, indicating more preferential tumoraccumulation with siRNA-L₂ relative to jetPEI NPs.

TABLE 2 Key pharmacokinetic parameter comparisons of siRNA-L₂ vs. jetPEINPs Parameter jetPEI NPs siRNA-L₂ (1) siRNA-L2 (10) P value_(L2(1)) Pvalue_(L2)(10) AUC_(tumor, 0.5-48 h), 1.48 × 10¹⁰  3.61 × 10¹⁰  2.90 ×10¹¹ radiance × h Liver:tumor ratio_(24 h, orthotopic) 2.6 ± 0.2 15.4 ±5.0 40.7 ± 5.2 0.1117 0.0007 Liver:tumor ratio_(24 h, PDX) 1.5 ± 0.4 7.8 ± 1.8 0.1357 Liver:tumor ratio_(48 h, orthotopic) 2.8 ± 0.2  6.2 ±2.6 43.1 ± 2.7 0.1739 0.0007 Fold tumor cell uptake, 30 min 7.2 ± 0.634.7 ± 4.7 325.2 ± 29.0 0.0001 <0.0001 Fold tumor cell uptake, 24 h 16.7± 1.0  31.0 ± 3.6 326.8 ± 16.0 0.0032 <0.0001

To further annotate the ability of siRNA-L₂ to avoid the typical MPSorgan accumulation of nanoparticles and accumulate preferentially withintumors, we evaluated the tumor:liver radiance ratio. At the 10-mg/kgsiRNA-L₂ dose, a tumor:liver accumulation of more than 40:1 was observedat both 24 and 48 h, indicating successful accumulation at a nonhepaticsite (FIGS. 5E-F and Table 2). In contrast, jetPEI NPs displayed atumor:liver ratio of below 3:1, a more than 15-fold decrease comparedwith siRNA-L₂ at 10 mg/kg and also lower than that observed for siRNA-L₂at 1 mg/kg (which achieved a tumor:liver ratio of ˜15:1).

The clear superiority of siRNA-L₂ in the orthotopic model motivatedinvestigation in a more clinically relevant patient-derived xenograft(PDX) model of triple-negative breast cancer. Dose-matched siRNA-L₂ andin vivo jetPEI NPs at 1 mg/kg were injected i.v. and biodistribution wasevaluated at 24 h. siRNA-L₂ attained 4.0-fold greater tumor distributionin the PDX model than jetPEI NPs (whereas there was a 2.2-fold increasedtumor delivery in the dose-matched orthotopic model) at 24 h (FIGS. 5G-Hand 8A). Compared with the orthotopic model, achieving tumoraccumulation in the PDX model was more challenging. The added challengeof PDX tumors was more detrimental for the tumor delivery with jetPEINPs than siRNA-L₂. Total tumor accumulation in PDX tumors was 4.3-foldlower than orthotopic tumors for jetPEI NPs whereas it was only reducedby 2.4 fold for siRNA-L₂. The lower MPS accumulation of siRNA-L₂relative to NPs was consistent in the PDX model, with siRNA-L₂ againshowing a marked improvement in tumor:liver ratio (8:1 vs. 1:1) (FIG.8B).

siRNA-L₂ Exhibits Homogeneous Distribution and High CellularInternalization at the Tumor Site. The small size of albumin-boundsiRNA-L₂ is expected to increase tissue penetration and homogeneity ofdistribution over nanoparticles. Using an in vitro tumor spheroid model,the penetration and distribution of siRNA-L₂ vs. jetPEI NPs throughout3D tumor architecture was evaluated. The siRNA-L₂ showed homogeneous andsubstantial cell uptake throughout the entirety of spheroids, whereasjetPEI NPs remained localized largely around the edges of the spheroid(FIG. 9A). Unmodified siRNA showed improved penetration into theinterstitial spaces compared with the jetPEI complexes, but exhibitedlower overall fluorescence than siRNA-L₂ (FIG. 10A). To complement theseresults, flow cytometry was used to measure uptake per cell (asquantified by mean intracellular fluorescence) in tumor spheroids thatwere dissociated and analyzed following siRNA formulation treatment. Thecellular internalization of siRNA-L₂ was twofold higher than that ofunmodified siRNA, evidencing an uptake benefit derived from hydrophobicmodification (FIG. 10B). Compared with jetPEI NPs, siRNA-L₂ exhibited agreater than fivefold uptake increase (FIG. 9B), with 84% ofsiRNA-L₂-treated cells positive for uptake compared with 27% ofjetPEI-NP-treated cells (FIGS. 10C-D).

These in vitro tumor spheroid results inspired an investigation of tumorpenetration and homogeneity of internalization by cells withinorthotopic breast tumors in vivo. Following i.v. injection of siRNA-L₂or jetPEI NPs, cells were isolated from excised tumors and evaluated byflow cytometry for cellular internalization. Tumor cells were identifiedby expression of green fluorescent protein (GFP). siRNA-L₂ outperformedjetPEI NPs at both 30 min and 24 h, with siRNA-L₂ at 1 mg/kg displaying5- and 2-fold increased uptake at respective time points and siRNA-L₂ at10 mg/kg showing 45- and 20-fold increased uptake (FIGS. 9C-D and Table2). At 30 min, mice treated with siRNA-L₂ at either dose displayeduptake in more than 96% of tumor cells, whereas jetPEI-NP-treated miceshowed uptake in only 60% of cells (FIG. 10E). The preferential andhomogeneous distribution of siRNA-L₂ to tumor sites and high uptake bytumor cells makes it ideally suited for cancer therapies.

siRNA-L₂ Elicits Sustained Silencing in an in Vivo Tumor Model. Thepromising tumor penetration characteristics of siRNA-L₂ inspiredexamination of its gene silencing efficacy in vivo in an orthotopicmouse tumor model. After treatment with luciferase-targeted siRNA orsiRNA-L₂ at days 0 and 1, luminescence was evaluated over 7 d, where anincrease in luminescence indicates tumor growth and successfulluciferase silencing abrogates the increase in luminescent signal.siRNA-L₂-treated tumors exhibited significantly reduced tumorluminescence in comparison with tumors treated with unmodifiedluciferase-targeting siRNA or inactive, control siRNA-L₂ sequences(FIGS. 9D and FIG. 11A). Comparing to the scrambled siRNA-L₂ control,maximum silencing was more than 60% at day 1, with nearly 50% silencingsustained at day 7, revealing the prolonged gene silencing capacity ofsiRNA-L2. Treatment with jetPEI NPs at a dose of 1 mg/kg elicitedsignificant (-30%) silencing at day 3, but silencing was fully abrogatedby day 7 (FIG. 11B). No change in mouse body weight was observed overthe course of treatment, further indicating that siRNA-L₂ treatment iswell-tolerated (FIGS. 11C-D).

As an initial proof-of-concept of activity against a therapeutically2Q:17 relevant gene, siRNA-L₂ that targets the negative regulator ofapoptosis, myeloid leukemia cell differentiation protein (MCL-1), wassynthesized. The siRNA-L₂ against MCL-1 achieved significant genesilencing in vitro at a reasonably low, 200-nM dose, and MCL-1 silencingcorrelated with a trend of functional increase in caspase activity(FIGS. 12A-B).

Discussion

Simple conjugation of a hydrophobic albumin-binding diacyl lipid moietyto siRNA is a powerful delivery strategy to improve siRNApharmacokinetic properties. L₂ conjugation increases circulationhalf-life, cellular internalization capacity, and tumor penetration andretention of siRNA while simultaneously reducing accumulation inclearance organs. These myriad benefits lead to enhanced and prolongedin vivo gene silencing in tumors, supporting siRNA-L₂ as a potentialcancer therapy that can act on currently undruggable targets.

Leveraging albumin as an endogenous nanocarrier is a relatively recentbut extremely promising strategy to extend the circulation persistenceof therapeutics. Clinically relevant examples range from Abraxane, analbumin-based nanoparticle that encapsulates Taxol, to Levemir, atherapeutic peptide modified to associate noncovalently with endogenousalbumin. siRNA, with its high potential medical impact butcharacteristically short circulation half-life, is an ideal candidate todevelop with albumin as an in vivo chaperone. Inducing high-affinitybinding of siRNA to albumin via modification with a lipidic moiety is alogical strategy. Previous work has shown siRNA amenable to lipidmodifications, which often confer improvements in nuclease resistanceand cellular internalization without impacting gene silencing.Conjugation with L₂ therefore has potential benefits on enhancingmolecule stability and uptake whereas also prompting in situ albuminbinding. Notably, this binding is noncovalent and dynamic. In itsphysiological role as a fatty acid carrier, albumin facilitates thecellular uptake of lipids, likely through a variety of mechanisms thatuse receptors for both albumin and lipid domains. Conjugation of L₂could allow siRNA-L₂ to hijack these natural pathways. Additionally, thehydrophobic interaction of the L₂ moiety with the cellular membranecould encourage siRNA-L₂ to be internalized independent of albumin.

L₂ modification as an albumin targeting approach is desirable forachieving pharmacokinetic improvements while maintaining simplicity andsafety. Despite the synthetic complexity of nano-particle systems,siRNA-L₂ possesses a circulation half-life above that ofnon-cross-linked polyion nanoparticles and nearly equivalent to thatobserved in a relatively intricate cross-linked micelle system usingcholesterol-modified siRNA. Perhaps more striking is the complete lackof toxicity observed for siRNA-L₂ at doses of 10 mg/kg, which sharplycontrasts with the reported toxicity and immunogenicity ofnanoparticulate carriers and our direct evaluation of in vivo jetPEI.siRNA-L₂ couples an improved circulation half-life with a lack ofdose-limiting side effects, and therefore is anticipated to enable verybroad therapeutic windows when developed against specific targets. Ourdemonstration of MCL-1 gene silencing and its link to caspase inductiondemonstrates the applicability of siRNA-L₂ to such therapeutic targets.Additionally, we expect that the efficacy of siRNA-L₂ could be furtheroptimized through modifications to enhance in vivo stability and throughidentification of siRNA sequences with extremely potent silencing.

Another associated challenge with nanoparticle delivery systems is theirpreferential accumulation within clearance organs, specifically theliver and spleen. Accumulation of synthetic and toxic/immunogenicnanoparticle components in these organs is the typical cause ofdose-limiting toxicities. However, siRNA-L₂ avoids capture in the MPSorgans, which is characteristic of nanoparticles, while also exhibitinga significant reduction in the rapid renal clearance associated withunmodified siRNA delivery. This is exemplified by the tumor:liveraccumulation ratio of more than 40:1 achieved by siRNA-L2. The disparitybetween in vivo jetPEI, with a ratio of less than 3:1, is pronounced.The lack of siRNA-L₂ retention in the liver is a key advantage overnanoparticulate delivery systems and will allow a greater percentage ofthe injected dose to be retained at its site of action in tumors. Thelower tumor:liver ratio observed with in vivo jetPEI and nanoparticlesystems in the literature is consistent with reported challenges inachieving efficient nanoparticle delivery to tumor sites; in acomprehensive analysis of nanoparticle delivery to solid tumors, themedian injected dose delivered to the tumor site was 0.7%. It is notablethat even in recent, advanced, and promising nanoparticle systems,including those that use modifications for “stealth” or targetingmechanisms, the ratio of tumor:liver accumulation is consistently closeto or below 1:1. The marked improvement of siRNA-L₂ in relative tumoraccumulation supports its translational promise.

There is also a significant tumor penetration benefit of siRNA-L₂ due toits small size relative to nanoparticle carriers. Whereas in vivo jetPEIdisplays poor penetration of tumor tissue, siRNA-L₂ distributeshomogeneously throughout tumor tissue and achieves consistently highuptake in tumor cells. The capacity of siRNA-L₂ to offer superior tumorpenetration is particularly significant given the highly inconsistentnature of clinical tumor vasculature and tissue morphology which limitsconsistent nanoparticle distribution. Here, we note that the PDX mousemodel is less permissive to delivery than the orthotopic model. PDXmodels are considered more clinically relevant, as they preserve thenative tissue architecture of the primary tumor through multiple in vivopassages and consistently recapitulate histopathologic and molecularcharacteristics, including drug responses and metastatic potential. Themore challenging nature of the PDX model relative to the orthotopicmodel [which is considered more stringent than the flank model] alignswith recent discussion suggesting that the permeable nature of commonlyused mouse tumor models has led to an overestimation of the EPR effect.Whereas nanocarriers like in vivo jetPEI may achieve efficacy in highlyvascularized or nonsolid tumors, they lack the ability to diffusethroughout the bulk of tumor architecture. Faced with a more difficultdelivery challenge in the PDX model, siRNA-L₂ maintains tumoraccumulation better than does in vivo jetPEI. As the majority of humansolid tumors contain regions of poor vascularization and displaydisparity in vessel permeability, the performance of siRNA-L₂ in the PDXmodel suggests applicability to a much broader range of cancers. Arecognition of the limitations of the EPR effect and a developingunderstanding of tumor heterogeneity calls for innovative solutions forsystemic RNAi cancer therapies. siRNA-L₂ deviates enormously from thestandard nanoparticle format, and its notable advantages should inspirefurther research into similar conjugate-based strategies.

In situ targeting of albumin as an endogenous carrier is a powerfulstrategy to enhance the bioavailability of siRNA and avoid the issuesassociated with synthetic nanocarriers. siRNA-L₂ surpasses conventionaldelivery systems in circulation persistence, safety, biodistributionprofile, and tumor penetration and cellular internalization. Ultimately,siRNA-L₂ achieves sustained gene silencing in tumors in vivo, providingstrong proof-of-concept for therapeutic efficacy. This work highlightsthe immense value of the siRNA-L₂ conjugate as a translational andpotentially transformative approach to improve i.v. RNAi cancertherapies.

Materials and Methods

Materials. Amine-modified single-stranded DNA (modification at 5′ end)or RNA (modification at 3′ end) and complementary single-stranded Cy5-,unmodified DNA, or unmodified RNA was obtained from Integrated DNATechnologies (for DNA) or GE Dharmacon. The pGreenFirel-CMV plasmid wasobtained from System Biosciences, and packaging plasmids pMDLg/pRRE,pRSV-Rev, and pMD2.G were purchased from Addgene. In vivo jetPEI waspurchased from VWR International.1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000] (DSPE-PEG2000-azide) was purchased from Avanti PolarLipids. NucBlue Fixed Cell ReadyProbes were purchased from LifeTechnologies. NAP-25 filtration columns were purchased from FisherScientific. RNeasy Mini Kit was purchased from Qiagen, iScript cDNASynthesis Kit from BioRad Laboratories, and Caspase-Glo 3/7 Assay fromPromega Corporation. All other reagents were purchased fromSigma-Aldrich.

Oligonucleotide-L₂ Synthesis. Single-stranded amine-modified oligo wasreacted with 10-fold molar excess ofdibenzocyclooctyne-PEG4-N-hydrox-ysuccinimidyl ester (DBCO-PEG₄-NHS)predissolved at 25 mM in DMSO. The reaction was carried out for 18 h atroom temperature at a 1 mM oligonucleotide concentration in 30% DMSO and70% PBS with 8 mM TEA. The product was diluted threefold in water andfiltered twice through NAP-25 columns, lyophilized, and then reactedwith fivefold molar excess of DSPE-PEG2000-azide for 24 h at a 0.1 mMoligonucleotide concentration in 50% methanol, 50% water. The reactionwas diluted and filtered one time through an NAP-25 column and thenpurified with reversed-phase HPLC using a Clarity Oligo-RP column(Phenomenex) under a linear gradient from 95% water (50 mMtriethylammonium acetate), 5% methanol to 100% methanol. The conjugatemolecular weight was confirmed using MALDI-TOF mass spectrometry(Voyager-DE STR Workstation) using 50 mg/mL 3-hydroxypicolinic acid in50% water, 50% acetonitrile with 5 mg/mL ammonium citrate as a matrix.The yield of the oligo-L₂ was quantified based on absorbance at 260 nm.The purified oligo-L₂ was annealed to its complementary strand togenerate Cy5-, unmodified DNA-L₂, or siRNA-L₂. Conjugation and annealingwas also confirmed via agarose gel electrophoresis.

DNA was used as a cost-effective analog for siRNA in imaging studies,and is referred to as siRNA/siRNA-L₂ in the example for simplicity andcohesion (except where the figure is intended to show a directcomparison between DNA and siRNA). DNA/siRNA and DNA-L₂/siRNA-L₂exhibited degradation on similar time scales (FIGS. 3E-F) and DNA-L₂exhibits similar albumin binding (FIG. 2A), validating its use as amodel for siRNA-L2.

Oligonucleotide-L₂ characterization. Critical micelle concentration ofoligo-L₂ was assessed fluorescently using Nile red, as describedpreviously. Briefly, different dilutions were prepared from a 1 mg/mLstock solution to obtain micelle samples ranging in concentration from0.0001 to 1 mg/mL Then, 10 μL of a 1 mg/mL Nile red stock solution inmethanol was added to 1 mL of each sample and incubated overnight in thedark at room temperature. The next day, samples were filtered with a0.45-μm syringe filter, and Nile red fluorescence was measured in96-well plates using a microplate reader (Tecan Infinite 500, TecanGroup Ltd.) at an excitation wavelength of 535±20 nm and an emissionwavelength of 612±25 nm. The CMC wasQ:20 defined, as previouslydescribed, as the intersection point on the plot of the Nile redfluorescence versus the copolymer concentration.

Degradation of siRNA and siRNA-L₂ was assessed by incubation in 60% FBSfor 4 h, 2 h, 1 h, 30 min, or 15 min and evaluation by agarose gelelectrophoresis with comparison with a control sample in water.

Evaluation of albumin binding to oligo-L₂ in vitro. PAGE gel migrationassay was used to assess binding of oligo-L₂ to BSA. 4-20% Mini-ProteanTGX Precast Q:21 Gel were run in the Tetra Blotting Module (BioRad).siRNA, siRNA-L₂, DNA, and DNA-L₂ were incubated with varying amounts ofBSA for 15 min. PAGE gels were stained using GelRed Nucleic Acid Stain(Biotium) according to manufacturer protocol and imaged under UV lightfor visualization of nucleic acid migration. Gels were subsequentlystained with Coomassie blue to evaluate BSA migration.

PAGE gel migration assay was used to assess binding of oligo-L₂ toalbumin in serum. siRNA or siRNA-L₂ was incubated with 9- or 13-foldmolar excess BSA or 50% or 75% FBS (for approximate matching of mass ofprotein loaded per well). siRNA and siRNA-L₂ were imaged under UV lightafter poststaining with GelRed. Serum proteins were stained withCoomassie blue.

Isothermal calorimetry (ITC) experiments were performed using a TAInstruments Nano ITC. Oligo-L₂ was prepared at a concentration of 0.1 mMand BSA was dissolved at a concentration of 0.25 mM from lyophilizedpowder in PBS. Titration experiments were carried out at 37° C. with a300-initial delay, 150-rpm stirring speed, and a sample cell volume(containing DNA-L₂) of 300 μL. Each injection was 2 μL, with aninjection interval of 180 s. Data were analyzed using an independentbinding site model with a blank constant correction incorporated toaccount for heat of dilution. All data analysis was performed in NanoITC software.

Cell Culture. Human epithelial breast cancer cells (MDA-MB-231) werecultured in DMEM (Gibco Cell Culture) supplemented with 10% FBS (Gibco)and 0.1% gentamicin (Gibco). Luciferase-expressing MDA-MB-231s weregenerated as previously described.

In Vitro Gene Silencing. MDA-MB-231s were treated with siRNA or siRNA-L₂complexed with in vivo jetPEI according to the manufacturer's protocol.The siRNA was either designed against the luciferase gene (luc siRNA) orwas a scrambled sequence (scr siRNA). Cells were seeded at 2,000cells/well in 96-well black-walled plates and allowed to adhereovernight. Cells were then treated in 10% serum for 24 h at a dose of100 or 50 nM siRNA. After 24 h, media was replaced withluciferin-containing media (150 μg/mL) before imaging with an IVISLumina III imaging system at 24 and 48 h.

To evaluate silencing of a therapeutically relevant gene, siRNAtargeting induced MCL-1 was used. MCF7 cells were treated withMCL-1-targeted or a scrambled control siRNA-L₂ at 200 nM in 10%serum-containing media for 24 h. At 48 h, RNA was harvested and MCL-1mRNA levels were evaluated using quantitative real time PCR. Inparallel, caspase activity was measured at 48 h using the Caspase Glo3/7 Assay (Promega) according to the manufacturer's protocol.

Evaluation of albumin binding to oligo-L₂ in vivo. Fluorescent(Cy-5-labeled) DNA and DNA-L₂ was injected into the tail vein of CD-1mice (4-6-wk-old, Charles Rivers Laboratories) at 1 mg/kg. Blood wascollected at 20 min postinjection, and serum was isolated. Serum frommice injected with DNA, DNA-L₂, or saline was evaluated via PAGE gelmigration assay was used to assess binding of oligo-L₂ to albumin invivo.

In vitro evaluation using tumor spheroids. MCF7 cells (ATCC) werecultured in DMEM supplemented with 1% penicillin-streptomycin and 10%FBS. Three-dimensional MCF7 spheroid cultures were established asdescribed pre-viously. Briefly, cells were grown to 50% confluence in 2Dculture. Cells were washed twice with trypsin (0.05%, Gibco), trypsinwas aspirated, and cells were incubated at 37° C. for 10-15 min. Cellswere resuspended in growth medium, pipetted to generate single-cellsuspensions, and counted (Bio-Rad TC20 Automated Cell Counter).Single-cell suspensions (7,500 cells per 500 μL) were seeded ineight-well chamber slides (Nunc Lab-Tek II) precoated with 10 μLgrowth-factor-reduced Matrigel (BD Biosciences) in growth mediacontaining 2% growth-factor-reduced Matrigel and cultured for 5 d. The8-well chamber slides were used for evaluation by confocal microscopy;the setup was scaled up to 12-well plates for flow cytometry and genesilencing studies and down to 96-well plates for cytotoxicity studies.

To evaluate tumor spheroid penetration by confocal microscopy, on day 5,cultures were treated with 100 nM Cy5-labeled DNA, DNA-L₂, or DNAcomplexed with in vivo jetPEI for 4 h in fresh growth medium. Cultureswere washed once with PBS and fixed for 2 min with BD Cyotfix/Cytopermsolution (BD Biosciences). After aspirating fixative and removingplastic chamber, cultures on slides were mounted with ProLong GoldAntifade with DAPI (Molecular Probes) and secured by coverslip. Slideswere stored at 4° C. before confocal imaging. Confocal imaging wasperformed using the Nikon Clsi+ system on a Nikon Eclipse Ti-OE invertedmicroscopy base. The PMT HV gain, laser power, and display settings wereset for maximal SNR based on control biological samples such thatnegative control samples lacking label had no background fluorescenceand treatment samples had no saturated pixels. Image acquisition andanalysis were performed using Nikon NIS− Elements AR version 4.30.01.

To evaluate tumor spheroid penetration by flow cytometry, on day 5,cultures were treated as described above. Cultures were washed once withPBS and tumor cells were dissociated from Matrigel for evaluation of Cy5fluorescence.

Blood Plasma Pharmacokinetics. Fluorescent (Cy-5-labeled) DNA and DNA-L₂were injected into the tail vein of CD-1 mice (4-6-wk-old, CharlesRivers Laboratories) at 1 mg/kg. Before injection, the mouse ear wasplaced on a coverslip on the Nikon Clsi+ confocal microscope system. Anartery within the ear was set in focus, and after injection, images ofthe artery were automatically collected every 2 s for 30 min. After 30min, animals were killed. Maximum initial fluorescence of the artery wasset to a time of 0 s. Artery fluorescence was evaluated by quantifying acircular ROI entirely within the vessel. Data were fit to a one-phaseexponential decay model (equation below) and half-life and area underthe curve were determined from these fits.

Fluorescence_(blood)=Fluoro₀ *e ^(−kt).

Biodistribution in Tumor-Bearing Mice. For the orthotopic mouse tumormodel, athymic nude female mice (4-6-wk-old, Jackson Laboratory) wereinjected in each mammary fat pad with 1×10⁶ MDA-MB-231 cells in DMEM:Matrigel (50:50). After 21 d, tumor-bearing mice were injected via thetail vein with 1 mg/kg (nucleic acid dose) of fluorescent DNA, DNA-L₂,or DNA loaded in in vivo jetPEI. After 30 min, 24 h, and 48 h, animalswere killed and the organs of interest (heart, lungs, liver, spleen,kidneys, and tumors) were excised. The fluorescence intensity in theorgans was quantified on an IVIS Lumina III imaging system at excitationwavelength of 620±5 nm and emission wavelength of 670±5 nm (n=3 animals,n=6 tumors). Tumor radiance data were fit to a one-phase exponentialdecay model (equation below), and area under the curve was determinedfrom these fits.

Radiance_(tumor)=Radiance₀ *e ^(−kt).

For the PDX mouse tumor model, the triple-negative line HCI-010 wastransplanted into one inguinal mammary fat pad (surgically cleared ofendogenous epithelium) of NOD-SCID (Jackson Laboratory) female mice ofQ:26 3-4 wk of age (64). After ˜8 wk, PDX tumors were harvested, cutinto 4 mm×2-mm pieces, serially transplanted into the cleared inguinalmammary fat pads of a new cohort of NOD-SCID female mice, and grown to avolume of 300-500 mm³. Tumor-bearing mice were injected via the tailvein with 1 mg/kg (nucleic acid dose) of fluorescent DNA-L₂ or DNAloaded in in vivo jetPEI. After 24 h, animals were killed, and theorgans of interest (heart, lungs, liver, spleen, kidneys, and tumors)were excised. The fluorescence intensity in the organs was quantified onan IVIS Lumina III imaging system at excitation wavelength of 620±5 nmand emission wavelength of 670±5 nm (n=2 animals, n=2 tumors).

Acute Toxicity in Liver and Kidney. CD31 mice were injected withsiRNA-L₂ (10 mg/kg) or in vivo jetPEI-loaded siRNA (1, 2 mg/kg). After24 h, blood was collected by cardiac puncture and then centrifuged at2,000×g for 5 min. Then, Q:27 plasma was harvested and tested by theVanderbilt Translational Pathology Shared Resource for systemic levelsof ALT, AST, BUN, and creatinine.

Tumor Distribution in Vivo After i.v. Injection. For the orthotopictumor model, athymic nude female mice (4-6-wk-old, Jackson Laboratory)were injected in each mammary fat pad with 1×10⁶ MDA-MB-231 cells inDMEM:Matrigel (50:50). After 21 d, tumor-bearing mice were injected viathe tail vein with saline, 1 or 10 mg/kg fluorescent DNA-L₂, or 1 mg/kgDNA loaded in in vivo jetPEI. Tumors were excised, and cells wereisolated from each tumor. A mixture of collagenase and DNase was used todissociate cells, and ammonium-chloride-potassium lysing buffer was usedto lyse red blood cells. Uptake of fluorescent DNA or DNA-L₂ wasevaluated by flow cytometry (n=4 animals, n=8 tumors). Tumor cells wereidentified as the cell population expressing GFP, whereas theGFP-negative cell population corresponded to native mouse cells.

Target Gene Silencing After i.v. Injection. Athymic nude female mice(4-6-wk-old, Jackson Laboratory) were injected in each mammary fat padwith 1×10⁶ MDA-MB-231 cells in DMEM:Matrigel (50:50). After tumorsreached a size of 50 mm², tumor-bearing mice were injected i.p. withluciferin substrate (150 mg/kg) and imaged for bioluminescence on anIVIS Lumina III imaging system 30 min postinjection. Next, the mice wereinjected via the tail vein with 10 mg/kg (based on siRNA dose) luc siRNAor siRNA-L₂ or an scr siRNA-L₂. Alternatively, mice were injected with 1mg/kg in vivo jetPEI complexed with luc or scr siRNA. Mice were imagedand treated at days 0 and 1 following treatment injection and imaged forbioluminescence over time. Relative luminescence was determined bymeasuring the raw luminescent intensity of each tumor on each day andcomparing to the initial signal at day 0 (n=10 tumors per group). Mousebody weight was evaluated at each of these time points to investigatetreatment toxicity.

Statistical Methods. The treatment groups were statistically comparedusing a one-way ANOVA test (for nonrepeated measures of more than twogroups) or a two-way ANOVA (for measures repeated at multiple timepoints) coupled with a Tukey means comparison test. For comparisonbetween two groups, an independent two-tailed t test was used. A P value<0.05 was deemed representative of a significant difference betweengroups. For all data shown, the arithmetic mean and SE are reported, andthe sample size (n) is indicated.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

What is claimed is:
 1. A compound comprising a RNA directly conjugatedto an albumin-binding group.
 2. The compound of claim 1, wherein thealbumin-binding group is hydrophobic.
 3. The compound of claim 1,wherein the albumin-binding group is hydrophobic and anionic.
 4. Thecompound of claim 1, wherein the albumin-binding group is a divalentlipidic moiety.
 5. The compound of claim 4, wherein the divalent lipidmoiety is a diacyl lipid.
 6. The compound of claim 4, wherein thedivalent lipidic moiety comprises1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000] (L₂).
 7. The compound of claim 1, wherein the RNA is siRNAor miRNA.
 8. The compound of claim 7, wherein the siRNA comprises afunctionalized siRNA.
 9. The compound of claim 8, wherein thefunctionalized siRNA is functionalized with a dibenzocyclooctyne moiety.10. The compound of claim 1, wherein the compound is complexed withalbumin.
 11. A compound comprising siRNA functionalized with adibenzocyclooctyne moiety and directly conjugated to1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000].
 12. A method of gene silencing, the method comprisingadministering a compound comprising a RNA directly conjugated to analbumin-binding group to a subject in need thereof.
 13. The method ofclaim 12, wherein the albumin-binding group is hydrophobic.
 14. Themethod of claim 12, wherein the albumin-binding group is hydrophobic andanionic.
 15. The method of claim 12, wherein the albumin-binding groupis a diacyl lipid.
 16. The method of claim 12, wherein thealbumin-binding group comprises1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethyleneglycol)-2000] (L₂).
 17. The method of claim 12, wherein the RNA is siRNAor miRNA.
 18. The method of claim 17, wherein the siRNA comprises afunctionalized siRNA.
 19. The method of claim 12, wherein theadministering comprises intravenous administration.
 20. The method ofclaim 12, wherein the method further comprises treating cancer in thesubject.